Cervical cancer is the second most common malignant tumor in female genital system. In 1949, human papilloma virus (HPV) particles were firstly discovered in wart leachate under electron microscopy by Sttauss. When Zur Hansen predicted that HPV might be the sexually transmitted carcinogenic factor in 1976, the relation between HPV infections and cervical cancers has aroused intense interest among researchers of the tumor pathogenesis. A large number of studies have convincingly proved that HPV was the main causative factor of cervical cancer, and could also cause other tumors in genital tract, mammary gland, digestive tract and respiratory tract. Recently, HPV infection rate in China has increased continuously, therefore, early prevention and treatment of cervical cancers has become extremely important.
The majority of women would be infected by genital HPV during the life time and the ratio of HPV-infected women is up to 10.4% worldwide. In most cases, HPV virus could be cleared by immune system within 1 to 2 years. However, chronic HPV infection could result in cervical intraepithelial neoplasia (CIN) such as CIN2 and CIN3, or even cervical cancer. According to previous reports, approximately 20% low-grade cervical lesions would develop into high-grade lesions and nearly 30% high-grade lesions would finally lead to malignant tumors. Therefore, early detection and prevention of HPV infection are very critical for decreasing mortality of cervical cancer and medical cost. At present, morphological test of exfoliated cervical cells by Pap smear acts as the main method for cervical cancer detection. However, the application of morphological test has been greatly limited by the high dependence on experts' experiences, difficulties in slide preparation, poor repeatability between inter-assay and intra-assay and high false-positive and false-negative rates, which usually result in low sensitivity and high rate of missed diagnosis. The positive detectable rate for early cervical cancer detection was only about 30% to 50%. In some areas, Pap smear test is replaced by liquid based cytology (LBC). LBC is a new semiautomatic or automatic technology for sample disposal, which is capable of analyzing sample automatically and providing remaining cells sample for other HPV detection. HPV DNA (e.g. HC2) detection is another wildly used molecular detection in clinic, which could assist cytological detection in identifying high-risk HPV virus. Even though HPV DNA detection is more sensitive than cell morphological detection, it still cannot distinguish continuous instantaneous infection from instantaneous infection. This is an extremely important factor for the transition of malignant tumor. Hence HPV DNA detection cannot be used to detect cancers because it is unable to reflect cancer starting (In the 2012 respective updated cervical cancers screening guidelines by America and China, exclusive use of HPV detection for screening cancers is not recommended in any age group).
HPV is a species-specific mucosa-infected virus, which belongs to small double-stranded circular DNA virus and contains about 8000 base pairs. The HPV virus contains 8 early open reading frames (E1-E8), 2 late open reading frames and one long non-coding regulatory region. In the early reading frames, E6 and E7 are the most important genes for stimulating cell growth. The study of Ziegent (2003) has confirmed that, HPV E6 and E7 genes are potential carcinogenic genes which are capable of cell transformation. The encoded E6 and E7 proteins are oncoproteins which can transform mouse epithelial cell in vitro and cause immortalization of human epithelial cell. Additionally, the persistent expression of HPV E6 and E7 proteins is necessary for maintaining immortalization in vitro. High-risk HPV E6 and E7 genes can induce cancer in nude mouse. Senung et al. (Seung H S, Jae k L, Oye S S. The relationship between cytokines and HPV-16, HPV-16E6, E7 and high-risk HPV viral load in the uterine cervix [J]. Gynecol Oncol, 2007, 104 (3):732-738) have reported that injection of HPV 16 E6 and E7 genes into mouse basal cells induces skin cancer. Therefore, the early-expression of E6 and E7 proteins of high-risk HPV is critical in carcinogenesis of cervical cancer. In carcinogenesis process, virus DNA is integrated into genome of a human cell. As control of E6 and E7 protein expression lacks, E6 and E7 are persistently expressed in epithelial cells of highly atypical cervical hyperplasia and cervical cancers. Consequently, HPV E7 protein can be a biomarker for high-grade cervical lesions and cervical cancer.
Nowadays, HPV L1 and some other auxiliary biological markers, such as p16INK4A, Ki67 and hTERT, are mainly used in HPV clinical immunohistochemical detection. There are three reasons why there is no suitable antibody for E7 protein detection. First, the low expression quantity of HPV protein in clinical tissues and cell samples requires that an antibody of high affinity. Second, HPV virus cannot survive in laboratory under standard tissue culture technique. Third, the immunosuppression activity of E7 protein results in poor immunoreaction in an animal which is challenged with use E7 protein. Moreover, the obtained antibodies are not specific to E7 proteins, because antibodies often have cross reaction with other HPV proteins.
Therefore, it is desired in the art to provide a convenient and reliable technique for detection of cancer with infection of high-risk HPV (especially cervical cancer) which is capable of identification of HPV type in the infected cancer cells.